Cultures were prepared from murine fore and hind limb buds of E11.25 to E11.75 embryos (obtained from breedings of homozygous male transgenic animals or C57Bl6F1/J males with C57Bl6F1/J females) as previously described with the following modifications (Cash et al. 1997). After proteolytic digestion, cells were filtered through a Cell Strainer (40 μm; Falcon) to obtain a single cell suspension. Culture media (40% Dulbecco's modified Eagle's medium and 60% F12 supplemented with fetal bovine serum to 10%; GIBCO BRL) was changed daily. BMP-2 or -4 (Genetics Institutes), AGN 194301 (Allergan Pharmaceuticals), and/or purified Xenopus Noggin protein was added to culture media at a concentration of 10 ng/ml, 1 μM, and 10 ng/ml, respectively. Addition/removal experiments included either adding or removing supplemented media on the indicated culture day. 24 h after culture initiation was considered day 1. To detect transgene-expressing cells, cultures were fixed and stained as previously described, with magenta-gal (BioSynth International Inc.) being substituted for X-gal. This was followed by alcian blue staining for cartilage-specific glycosaminoglycans (Lev and Spicer 1964). Alcian blue staining of magenta-gal–stained cultures turned the red precipitate to a purple color, as a result of incubating magenta-gal–stained cells at pH 1. This double-staining technique enables transgene-expressing cells to be localized with respect to alcian blue–stained cartilage nodules. Images were captured using a Sony DXC-950 3CCD color video camera and analyzed using Northern Eclipse image analysis software (Empix Imaging, Inc.) and composite figures were generated in CorelDraw.